SUMMARY Nearly 88,000 people die from alcohol-related causes annually, making it the fourth leading preventable cause of death in the United States. Understanding the molecular mechanisms underlying the neurological changes that lead to alcohol dependence is crucial for developing new, effective alcohol use disorder (AUD) treatments. Towards this goal, recent studies have identified genome-wide DNA methylation (DNAm) signals distinguishing alcoholic and non-alcoholic post-mortem brain tissue. In addition, a subset of the differential DNAm (D-DNAm) signals was associated with the altered expression of nearby genes, suggesting that DNAm regulates the gene expression patterns associated with alcoholic dependence. However, it is unknown whether some of the DNAm signals detected in the post-mortem tissue were preexistent, potentially contributing risk for hazardous drinking, rather than being induced by chronic alcohol. This study aims to distinguish these two possibilities, and to pinpoint the functional consequences of alcohol-induced DNAm in the prefrontal cortex (PFC). The study makes use of the highly relevant and well-characterized nonhuman primate (NHP) alcohol self- administration model. In this model, voluntary alcohol consumption levels are measured in rhesus macaques daily for ~12 months, enabling the accurate classification of natural ?low? and ?heavy? alcohol drinkers. In addition, a small portion of the PFC is biopsied prior to alcohol access, providing opportunity to establish baseline measures of DNAm. Genome-wide bisulfite sequencing (GWBS) will be used to identify alcohol-dose dependent, differentially methylated cytosines (DMCs) and regions (DMRs) in the PFC of the rhesus macaques. The same approach will be used to compare DNAm in PFC obtained from the macaques prior to alcohol access, enabling the detection of preexistent, alcohol-dose associated DNAm signatures. In addition, the macaque D-DNAm signals will be compared to D-DNAm data generated from post-mortem human alcoholic PFC tissue, identifying alcohol-associated DNAm that is replicated across two primate species. Next, the expression of DMC/DMR-associated genes, including both whole gene and alternative transcript expression, will be correlated with the DNAm data. Based on support from the human-macaque DNAm comparison, and corresponding expression data, a subset of compelling, novel gene targets will be selected for functional study. Target gene activity will be modulated using pharmacological and gene expression modification approaches in a murine model. The neural effects of target modulation will be evaluated using patch-clamp electrophysiological analysis, and alcohol use will be evaluated using intermittent alcohol, 2 bottle choice models. In total, this work will identify preexistent and alcohol-induced DNAm modifications in the primate PFC and will clarify the role of a subset of DNAm-linked genes in promoting alcohol use. The findings of this study will provide functional support for the design of promising new AUD treatments.